Autophagy Plays A Protective Role In Advanced Glycation End Products Induced Apoptosis Of Chondrocytes

ObjectiveOsteoarthritis?OA?is a chronic disabling disease characterized by the disintegration of articular cartilage,the destruction of cartilage tissue and the hyperplasia of the bone around the joints,and the age is a major risk factor for OA.Recent studies have shown that excessive apoptosis of chondrocytes and degradation of extracellular matrix paly important roles in the pathogenesis of OA.Advanced glycation end productwas produced through a nonenzymatic reactionbetween reducing sugars and free amino groups of proteins,lipids,or nucleic acids.It has longbeen assumed that nonenzymatic glycation is initiatedsolely by the spontaneous condensation of reducing sugarslike glucose with free amino groups in lysine or arginine residues.After formation of a covalent bondbetween the sugar and the amino acid,subsequent reactionsgive rise to the formation of AGEs.Autophagy is an essential process important for cell survival and homoeostasis.Autophagy involves the formation of a membrane around a region of the cytoplasm,sequestering macromolecules like proteins and organelles,and the fusion of the resultant vesicle with a lysosome in which the contents are degraded.Through autophagy starving cells degrade materials within their own cells to provide necessary nutrients for more essential processes.Studies discovered that advanced glycosylation end products accumulated in longevity protein increased susceptibility of osteoarthritis.other studies discovered that Autophagy palys a protective role in the pathogenesis of OA.The common effects advanced glycosylation end products and autophagy in OA has been rarely reported..In this paper,we will study the effect of advanced glycosylation end product on chondrocytes and the effect of autophagy on advanced glycation end product induced apoptosis in chondrocytes.MethodsThe first part is study the effect of advanced glycation end product on chondrocytes.Chondrocytes were separated from cartilage of five patients with osteoarthritis carried total knee replacement surgery,and chondrocytes were cultured in DMEM/F12 supplemented with 10%FBS,The cells were identified by observing cell morphology under phase-contrast microscope,drawing growth curve,toluidine blue staining and type II collagen immunohistochemistry staining.The optimal stimulation time and the optimal stimulus concentration of advanced glycation end product on chondrocytes were selected by the preexperiment.The effects of advanced glycation end product on the activity of chondrocytes were determined using MTT assay.The effects of advanced glycation end product on the expression of tumor necrosis factor-alpha?TNF-??and nuclear factor-kappa B?NF-?B?was detected by quantitative real-time polymerase chain reaction?qRT-PCR?.The effects of advanced glycation end product on the reactive oxygen species?ROS?production was determined by fluorescent probe.The effects of advanced glycation end product on theapoptosis of chondrocytes were determined byAnnexin V-FITC/PI.The second part is study the effect of autophagy on the advanced glycation end product induced theapoptosis of chondrocytes.Chondrocytes were separated from cartilage of five patients with osteoarthritis carried total knee replacement surgery,and chondrocytes were cultured in DMEM/F12 supplemented with 10%FBS,The cells were identified by observing cell morphology under phase-contrast microscope,drawing growth curve,toluidine blue staining and type II collagen immunohistochemistry staining.The chondrocytes of the untreated group was administered with 100ug/ml BSA in the medium during the culture for 12h.The chondrocytes of the AGEs group was administered with 100ug/ml AGE-BSA in the medium during the culture for12h.The chondrocytes of the AGEs+rapamycin group was pretreated with 1mmol rapamycin for2h before AGE-BSA incubation.The cell viability was determined using MTT assay.The expression of tumor necrosis factor-alpha?TNF-??and nuclear factor-kappa B?NF-?B?was detected by quantitative real-time polymerase chain reaction?qRT-PCR?.The reactive oxygen species?ROS?production was detected by fluorescent probe.The apoptosis of chondrocytes were determined by Annexin V-FITC/PI.ResultsThe first part is study the effect of advanced glycation end product on chondrocytes.Comprehensive analysis the growth curve of chondrocytes,toluidine blue staining and collagen type II immunohistochemical staining results that after four generations the cartilage cell proliferation after falling growth curve is flat,and amino polysaccharide cell secretion and synthesis of collagen type II ability also declined significantly,so this study choose chondrocytes in 2-4 generation for experiments.The optimal stimulation time of advanced glycation end product on chondrocytes was 100ug/ml,and the optimal stimulus concentration of advanced glycation end product on chondrocytes was 12 hours.The chondrocytes of the untreated group was administered with 100ug/ml BSA in the medium during the culture for 12h.The chondrocytes of the AGEs group was administered with 100ug/ml AGE-BSA in the medium during the culture for 12h.Advanced glycation end product can significantly decrease the activity of chondrocytes?P<0.001?.Advanced glycation end product can significantlyincreased the expression of TNF-??P<0.001?,and advanced glycation end product can significantlyincreased the expression of NF-?B?P<0.001?.Advanced glycation end product can significantlyincreasedintracellular reactive oxygen species production and accumulation?P<0.001?.Advanced glycation end product can significantlyaccelerated chondrocytes apoptosis?P<0.001?.The second part is study the effect of autophagy on the advanced glycation end product induced theapoptosis of chondrocytes.The MTT was used to detected the activity of chondrocytes,compared with the untreated group,the A₄₉₀ of AGEs group and AGE+rapamycin group at12hours was significantly decreased?P<0.001?.Compared with the AGEs group,the A₄₉₀ of AGE+rapamycin group at 12 hours was significantly increased?P=0.010?,indicating autophagy can protects advanced glycation end product induced the damage of chondrocytes.Cellular LC3 II protein expression was measured by western blot to present the level of autophagy.The LC3-II expression in chondrocytes treated with AGEs adding rapamycin was much higher than that in chondrocytes without treatment and AGEs treatment?both P<0.001?.There was no significance between chondrocytes with AGEs treatment and chondrocytes without treatment.The effects of AGEs and autophagy on the expression of TNF-?and NF-?B were detected by RT-PCR,AGEs adding rapamycin treatment,the RNA expression levels of TNF-?and NF-?B in chondrocytes were reduced compared to single AGEs incubation.When the chondrocytes were pretreated with rapamycin before AGEs incubation,the average fluorescence absorbance of cellular ROS production was lower than that with AGEs incubation?P<0.01?.To evaluate the apoptosis of chondrocytes under autophagy,the chondrocytes were stained by Annexin V-FITC/propidium iodide?PI?and detected byflow cytometer,with the rapamycin pretreatment,the rate of apoptosis significantly decreased under the autophagy protection?P<0.01?.ConclusionsThis study established a two-step enzyme digestion separation of chondrocytes,primitive and subculture of chondrocytes,toluidine blue staining and type II collagen immunohistochemical staining method for identification of chondrocytes,and through the comprehensive analysis of growth curve,toluidine blue staining and collagen type II immunohistochemical staining results determine the chondrocytes test generation.Advanced glycosylation end products by reducing the activity of chondrocytes,increased the expression of TNF-?and NF-?B,increasedintracellular reactive oxygen species production and accumulation,accelerated chondrocytes apoptosis to damage the chondrocytes.Autophagy ameliorates the AGEs-induced adverse effects by inhibition of chondrocytes activity decreased,inhibition the increased the expression of TNF-?and NF-?B,inhibition the increasedintracellular reactive oxygen species production and accumulation,and inhibition the accelerated chondrocytes apoptosis.